CRISPR/CasRx-Mediated RNA Knockdown Reveals That ACE2 Is Involved in the Regulation of Oligodendroglial Cell Morphological Differentiation

Abstract

Angiotensin-converting enzyme 2 (ACE2) plays a role in catalyzing angiotensin II conversion to angiotensin (1–7), which often counteracts the renin-angiotensin system. ACE2 is expressed not only in the cells of peripheral tissues such as the heart and kidney, but also in those of the central nervous system (CNS). Additionally, ACE2 acts as the receptor required for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), whose binding leads to endocytotic recycling and possible degradation of the ACE2 proteins themselves. One of the target cells for SARS-CoV-2 in the CNS is oligodendrocytes (oligodendroglial cells), which wrap neuronal axons with their differentiated plasma membranes called myelin membranes. Here, for the first time, we describe the role of ACE2 in FBD-102b cells, which are used as the differentiation models of oligodendroglial cells. Unexpectedly, RNA knockdown of ACE2 with CasRx-mediated gRNA or the cognate siRNA promoted oligodendroglial cell morphological differentiation with increased expression or phosphorylation levels of differentiation and/or myelin marker proteins, suggesting the negative role of ACE2 in morphological differentiation. Notably, ACE2′s intracellular region preferentially interacted with the active GTP-bound form of Ras. Thus, knockdown of ACE2 relatively increased GTP-bound Ras in an affinity-precipitation assay. Indeed, inhibition of Ras resulted in decreasing both morphological differentiation and expression or phosphorylation levels of marker proteins, confirming the positive role of Ras in differentiation. These results indicate the role of ACE2 itself as a negative regulator of oligodendroglial cell morphological differentiation, newly adding ACE2 to the list of regulators of oligodendroglial morphogenesis as well as of Ras-binding proteins. These findings might help us to understand why SARS-CoV-2 causes pathological effects in the CNS.

Investigating the Protective Effects of a Citrus Flavonoid on the Retardation Morphogenesis of the Oligodendroglia-like Cell Line by Rnd2 Knockdown

“…sc-35425; Santa Cruz, CA, USA). Control ( Photinus pyralis luciferase) siRNA was described previously [ 31 ].…”

Knockdown of Rab7B, But Not of Rab7A, Which Antagonistically Regulates Oligodendroglial Cell Morphological Differentiation, Recovers Tunicamycin-Induced Defective Differentiation in FBD-102b Cells

“…2.3. Cell culture, differentiation, and transfection FBD-102b cells (provided by Dr. Tomo-Oka, Tokyo University of Science), which belong to an oligodendroglial precursor cell line [17][18][19][20], were cultured on normal cell culture dishes (Nunc brand, Thermo Fisher Scienti c, Waltham, MA, USA) in Dulbecco's modi ed Eagle's medium (DMEM)/F-12 mixed medium (Nacalai Tesque) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco brand, Thermo Fisher Scienti c) and penicillin and streptomycin mixed solution (Nacalai Tesque) in 5% CO 2 at 37°C. Cells were transfected with the respective synthesized 21-mer siRNAs with dTdT using the ScreenFect siRNA transfection kit (Fuji lm, Tokyo, Japan) in accordance with the manufacturer's instructions.…”

“…The medium was replaced 4 hours after transfection and cells were further cultured for experiments. Less than 5% of attached cells contained incorporated trypan blue (Nacalai Tesque) at 48 hours after transfection [17][18][19][20].…”

“…To induce differentiation, cells were cultured on polylysine (Nacalai Tesque)-coated cell culture dishes (Greiner Bio-One, Oberösterreich, Austria) in culture medium without FBS until 5 days in 5% CO 2 at 37°C [17][18][19][20]. Cells with no primary process were classi ed as category 1; cells with primary processes were classi ed as category 2; and cells with secondary processes branched from primary processes or with myelin membrane-like widespread processes were classi ed as category 3 [17][18][19][20]. Category 2 corresponded to these intermediate phenotypes.…”

“…If so, how Rab7A and Rab7B regulate differentiation needs to be determined. Herein we describe that Rab7A and Rab7B differentially regulate morphological differentiation in FBD-102b cells, which are models of differentiation of oligodendroglial precursor cells [17][18][19][20]. It is of note that knockdown of Rab7B greatly promoted differentiation.…”